G protein-coupled receptors (GPCRs) play a central role in a multiplicity of physiological processes. It is assumed that in the human genome about 1000 genes code for this receptor family. Approximately 60% of the pharmaceuticals presently available through prescription act as GPCR agonists or antagonists. This underlines the importance of this receptor class for the pharmaceutical research industry. Owing to the size and importance of said protein family and in view of the fact that physiological ligands are still unknown for many GPCRs (orphan GPCRs), it can be assumed that this receptor class will be one of the most important reservoirs for suitable target proteins in the search for novel medicinal substances in the future.
GPCRs are a family of integral membrane proteins which are located on cell surfaces. They receive signals from extracellular signaling substances (e.g. hormones, neurotransmitters, peptides, lipids) and transfer these signals into the cell interior via a family of guanine nucleotide-binding proteins, the “G proteins”. Depending on the receptor specificity, the G protein activated and the cell type, these receptors induce various signal transduction pathways.
All GPCR polypeptide chains fold into seven α-helices which span across the phospholipid bilayer of the cell membrane. The seven membrane passages result in the formation of extra- and intracellular loops which allow extracellular ligand binding and intracellular coupling of G proteins. For this reason, GPCRs are also denoted seven-pass transmembrane receptors.
All G protein-coupled receptors act according to a common basic pattern: binding of an extracellular ligand leads to a conformational change in the receptor protein which enables the receptor protein to contact a G protein. G protein-mediated signal transduction cascades in the cell finally lead to a biological response of the cell.
G proteins are heterotrimeric proteins which consist of the subunits α, β and γ. They are located on the inside of the cell membrane via lipid anchors. Coupling of activated GPCRs to G proteins induces a GDP/GTP exchange at the Gα subunit and dissociation of the heterotrimeric G protein into an α and a βγ subunit. Both the activated α subunit and the βγ complex are able to interact with intracellular effector proteins.
Activation of membrane-bound adenylate cyclase (AC) by Gαs-type G proteins, for example, leads to an increase in the intracellular cAMP level or, in the case of activation by Gαi-type G proteins, to the decrease therein. Gq-type G proteins activate phospholipase C (PLC) which catalyzes the formation of inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). These molecules lead to the release of Ca2+ from intracellular storage organelles or to activation of proteinkinase C (PKC).
The polynucleotide sequence and the amino acid sequence of the human EDG2 (Endothelial Differentiation Gene 2) has been made available to the public. The sequence is available for example from NCBI (Accession: NM—001401). The protein sequence is available from Swiss Prot (Accession: Q 92633). Cloning of the receptor from a human lung cDNA library was published in “An et al., Biochem. Biophys. Res. Commun. 24, 231 (1997)”.
The full length sequence encodes a 359 amino acid protein which belongs to the superfamily of guanine nucleotide-binding protein-coupled receptors (GPCR). Human EDG2 mRNA is widely distributed in human tissues with the highest abundance in brain. HEK293 cells expressing the human EDG2 protein showed an elevated response to lysophosphatidic acid (LPA) in a serum response element reporter gene assay, which was LPA concentration dependent and specific to LPA. The mouse counterpart of EDG2 protein was also identified as a receptor for LPA.
Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are potent phospholipid mediators with diverse biological activities. Their appearance and functional properties suggest possible roles in development, wound healing, and tissue regeneration. The growth-stimulating and other complex biological activities of LPA and S1P are attributable in part to the activation of multiple G protein-mediated intracellular signaling pathways. Several heterotrimeric G proteins, as well as Ras- and Rho-dependent pathways play central roles in the cellular responses to LPA and S1P.